A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog

Significance Sonic Hedgehog (Shh) is a key signaling molecule that plays important roles in embryonic patterning, cell differentiation, and organ development. Although fundamentally important, the molecular mechanisms that regulate secretion of newly synthesized Shh are still unclear. Our study reveals a role for the cargo receptor, SURF4, in facilitating export of Shh from the endoplasmic reticulum (ER) via a ER export signal. In addition, our study provides evidence suggesting that proteoglycans promote the dissociation of SURF4 from Shh at the Golgi, suggesting a SURF4-to-proteoglycan relay mechanism. These analyses provide insight into an important question in cell biology: how do cargo receptors capture their clients in one compartment, then disengage at their destination?

or Leica STED TCS SP5 II Confocal Laser Scanning Microscope (Leica, Germany). To quantify the colocalization between ERGIC53 and GM130, the juxta-nuclear area labelled by GM130 was outlined manually using the freehand selections function in Fiji as the region of interest (ROI). To quantify the colocalization between SURF4-HA and GM130 or ERGIC53, the juxta-nuclear area labelled by SBP-EGFP-ShhN was outlined manually in Fiji as the ROI. To quantify the colocalization between ShhN and GM130, the juxta-nuclear area labelled by ShhN was outlined manually in Fiji as the ROI. All of the pixels in the ROI were not saturated. The Pearson's R value (no threshold) in the ROI between two channels was then calculated using Coloc 2 in Fiji.
For CRISPR experiments, single-guide RNA (sgRNA) sequences ligated into pX458 (pSpCas9 BB-2A-GFP) plasmids were purchased from GenScript. Transfections were performed with TransitIT-293 (Mirus Bio) per manufacturer's instructions. Clonal cell lines were derived by diluting cell suspensions to a single cell per well and expanding individual wells. Genotyping of clonal cell lines was performed by Sanger sequencing of target site PCR amplicons of genomic DNA isolated by Puregene kit (Quiagene). sgRNAs were as follows: SURF4, 5'AGTCGCGCTGCTCGCTCCAC3' targeting exon 1. The cells were collected and lysed by HKT buffer (100 mM KCl, 20 mM Hepes, pH 7.2, 0.5% Triton X-100). The bound proteins and cell lysates were analysed by immunoblotting.

Retention Using Selective Hook (RUSH) assay, antibody uptake assay and permeabilized cell assay
For antibody uptake assays, HeLa cells were treated (or not) with 2.5 mM xyloside in complete medium.
24hr after xyloside treatment, cells were transfected with plasmids encoding Str-KDEL_SBP-EGFP-HA-Shh FL . On day 2 after xyloside treatment, cells were incubated without biotin or with 40 μM biotin and 100 ng/μl cycloheximide in complete medium for 1 hr. After incubation, mouse anti-HA antibodies were added to the incubation medium at a 1:200 dilution to label the ShhN fusion construct that had been delivered to the cell surface. After an additional incubation for 40min at 37 ℃, cells were fixed for 15 min with 4% paraformaldehyde in PBS and then a standard immunofluorescence procedure was performed using rabbit anti-GFP antibodies as the primary antibodies.
Permeabilized cell assay was performed as described previously (3). Briefly, HeLa cells transfected with Str-KDEL_SBP-EGFP-ShhN were treated with 0.04 mM biotin at 37 °C for 4 min, followed by three washes in cold KOAc buffer (110 mM KOAc, 2 mM Mg(OAc)2, 20 mM Hepes, pH 7.2). Then cells were permeabilized by 0.03 mg/ml digitonin in KOAc buffer for 6 min at room temperature. The permeabilized cells were washed with cold KOAc buffer. After 5 min of incubation on ice with cold 0.5 M KOAc buffer followed by three washes in cold KOAc buffer to remove cytosolic proteins, the permeabilized cells were then incubated at 37 °C for 15 min in KOAc buffer containing 2 mg/ml rat liver cytosol, 0.04 mM biotin, 500 μM GDP/GTPγS, and an ATP regeneration system (40 mM creatine phosphate, 0.2 mg/ml of creatine phosphokinase, and 1 mM ATP). The cells were then washed with cold KOAc buffer, fixed, and stained with specific antibodies.

Immunoprecipitation, protein purification, and binding assay
Immunoprecipitation of HA-tagged ShhN was performed by incubating 200 µl of 0.5 mg/ml cell lysates from HEK293T cells transfected with ShhN-HA in HKT buffer with 10 μl of compact anti-HA agarose affinity beads with mixing at 4 °C overnight. After incubation, the beads were washed 4 times with 1 ml of HK buffer (100 mM KCl, 20 mM Hepes, pH 7.2), and the bound material was analyzed by Coomassie blue staining and immunoblotting.
Binding assays between Myc-tagged SURF4 or Myc-tagged SURF4 ED-AA and HA-tagged ShhN or ShhN ∆33-39 were performed by treating HEK293T cells co-transfected with plasmids encoding the indicated proteins in 1 × PBS containing 2 mM dithiobis[succinimidylpropionate] (DSP) and 2 mM CaCl2 at room temperature for 30 min, and then quenched with 25 mM Tris-HCl, pH 7.5. 200 µl of 0.5 mg/ml cell lysates were incubated with 10 μl of compact anti-HA agarose affinity beads with mixing at 4 °C overnight. After incubation, the beads were washed 4 times with 1 ml of HK buffer (100 mM KCl, 20 mM Hepes, pH 7.2), and the bound material was analyzed by immunoblotting.
Purification of GST-tagged ShhN 25-49 and GST-tagged SURF4 49-60 was performed as described previously (4). GST pull-down assays were carried out with 10 μl of compact GSH beads bearing around 5 µg of GST-tagged ShhN 25-49 . The beads were incubated with 200 µl of 0.5 mg/ml of cell lysates from HEK293T cells transfected with HA-SURF4 in HKT buffer at pH 6.0 or 7.2 with mixing at 4 °C overnight.
After incubation, the beads were washed three times with 500 µl of HKT buffer and twice with 500 µl of HK buffer, and the bound material was analyzed by immunoblotting.
After incubation, 15 ml buffer containing around 5 µl beads containing 5 nmol of peptides was added to the reaction mixture for 1 h at 4 °C. The beads were washed four times with 500 µl of HK buffer and analyzed by immunoblotting.

Label-free quantitative mass spectrometry
Mass spectrometry was performed to identify the proteins involved in Shh secretion. After transfecting plasmids encoding Shh-HA or IGF2-HA into HEK293T cells, the immunoprecipitation was processed, and the bound proteins were analyzed by Coomassie Blue (Bio-Safe TM Coomassie-G250) staining. The protein gel was cut into small fragments and washed with 25 mM NH4HCO3/50% acetonitrile at room temperature for 15 min for three times. Then the gel fragments were shrunken by acetonitrile at room temperature for 15 min and dried by speed vacuum. The dried protein gel pieces were reduced by 0.1 M NH4HCO3 containing 10 mM TCEP at 55 °C for 45 min and alkylated by 0.1 M NH4HCO3 containing 55 mM Indoacetamide at room temperature in the dark for 45 min. After that, the gel pieces were washed by 0.1 M NH4HCO3 and repeated the steps of shrink and dry. Then the proteins were digested by 50 mM NH4HCO3 containing 20 ng/μl sequencing grade modified trypsin (Promega, number V511A) on ice for 45 min and incubated in 50 mM NH4HCO3 at 37 °C overnight. 25 mM NH4HCO3 and 60% acetonitrile containing 5% formic acid were used to exact the peptides respectively. Then the samples were dried with speed vacuum. The dried peptides were dissolved into 0.1% trifluoroacetic acid to remove the surfactant, desalted using pierce C18 spin column and dried by speed vacuum. Finally, the resulted peptides were analyzed by Mass Spectrometer. The tandem mass spectra were then subject to protein identification and label-free quantification by Proteome Discoverer.
The proteins that were associated with the Shh or IGF2 were identified by comparing the peak intensity of the identified protein in the experimental group with the control group.

In vitro vesicle formation assay
In vitro vesicular release assays were performed as described previously (2, 5). Briefly, Day 1 after transfection with plasmids encoding HA-tagged different version of ShhN, HEK293T cells grown in one 10cm dish at around 90% confluence were permeabilized in 3 ml of ice-cold KOAc buffer containing 40 µg/ml digitonin on ice for 5 min, and the semi-intact cells were then sedimented by centrifugation at 300 × g for 3 min at 4 °C. The cell pellets were washed twice with 1 ml of KOAc buffer and resuspended in 100 µl of KOAc buffer. The budding assay was performed by incubating semi-intact cells (around 0.02 OD/reaction) with 2 mg/ml of rat liver cytosol in a 100 µl reaction mixture containing 200 µM GTP and an ATP regeneration system in the presence or absence of 0.5 µg of Sar1A (H79G). After incubation at 32 °C for 1 h, the reaction mixture was centrifuged at 14,000 × g to remove cell debris and large membranes. The medium-speed supernatant was then centrifuged at 100,000 × g to sediment small vesicles. The pellet fraction was then analyzed by immunoblotting. For density gradient flotation assays, the pellet fraction was resuspended in 100 µl of 35% OptiPrep and overlaid with 700 µl of 30% OptiPrep and 30 µl of KOAc buffer.
The samples were centrifuged at 55,000 rpm in a TLS55 rotor in a Beckman ultracentrifuge for 2 hr at 4 °C.
After centrifugation, fractions were collected from the top to the bottom of the tube, and the top fraction was analyzed by SDS-PAGE and immunoblotting.

Movie 1
A time-lapse video of SBP-EGFP-ShhN in HeLa cells untreated with xyloside. This video was generated from a time-lapse series of confocal images of SBP-EGFP-ShhN following biotin addition at an interval of 30 seconds. Representative frames are shown in Figure S10.

Movie 2
A time-lapse video of SBP-EGFP-ShhN in HeLa cells untreated with xyloside. This video was generated from a time-lapse series of confocal images of SBP-EGFP-ShhN following biotin addition at an interval of 30 seconds. Representative frames are shown in Figure S10.

Movie 3
A time-lapse video of SBP-EGFP-ShhN in HeLa cells treated with 2.5 mM xyloside. This video was generated from a time-lapse series of confocal images of SBP-EGFP-ShhN following biotin addition at an interval of 30 seconds. Representative frames are shown in Figure S10.

Movie 4
A time-lapse video of SBP-EGFP-ShhN in HeLa cells treated with 2.5 mM xyloside. This video was generated from a time-lapse series of confocal images of SBP-EGFP-ShhN following biotin addition at an interval of 30 seconds. Representative frames are shown in Figure S10.